Detection and quantitation of a specific nucleic acid sequence present in a sample is a known diagnostic method with great specificity. This specificity is based on the knowledge of the specific sequence and the generation of probes which are specific and complementary.
Methods for detection and quantitation of specific nucleic acid sequences are illustrated by the following patents: (1) U.S. Pat. No. 5,130,238 is directed toward an improved process for amplifying a specific nucleic acid sequence. The improvement of the amplification process involves the addition of dimethylsulfoxide (DMSO) alone or in combination with bovine serum albumin (BSA); (2) U.S. Pat. No. 4,683,195 is directed toward a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof; (3) U.S. Pat. No. 4,683,202 is directed toward a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof; (4) U.S. Pat. No. 4,486,539 is directed toward a method for identifying nucleic acids by a one-step sandwich hybridization test; and (5) WO 91/02814 is directed toward a process for amplifying a specific nucleic acid sequence.
The use of highly specific nucleic acid probes is in some cases the only method which can yield accurate results when the protein is absent, such as is the case for analysis of genetic defects such as cystic fibrosis. It is also valuable in the case of a latent viral infection such as HIV1 or herpes where little or no protein is produced by the infection. The great specificity of the nucleic acid probes also makes them valuable in the diagnosis of infectious agents which are difficult to identify with antibodies due to cross reaction and lack of cross reaction between isotypes of these agents. In addition, the analysis of DNA sequences allows the rapid and effective selection of a probe which will be specific. This is not possible with antibody-based reagents.
The greatest difficulty and limitation with applying existing nucleic acid probe technology is the complexity and slow methodology for the detection of specific sequences. With the amplification of nucleic acids, the limitations of nucleic acid probe methods associated with low levels of target molecules have been solved in U.S. Pat. Nos. 5,130,238; 4,683,195; 4,683,202, identified above.
The use of natural amplification has been used in certain cases to remove this problem. This is exemplified by the use of ribosomal RNA with up to 100,000 copies per cell as taught in U.S. Pat. No. 4,851,330. This method, in order to be effective without the need to culture the infectious agent, makes use of a rapid chemiluminescence detection system which requires a number of incubations and washes. This method, however, is also limited only to selected cellular pathogens and is of no use in the case of viral or genetic defects.
Notwithstanding the amplification processes disclosed in the prior art, the present invention requires no pretreatment of the sample such as binding to solid phases or membranes, denaturing of the sample, purification of the sample by extraction of oil, protein or by gel electrophoresis and the probes can be added directly to the amplification mixture hybridized and analyzed. This was surprising in the light of the potential that the probe sequences would be modified or cause sample destruction mediated by the enzymes and conditions in the amplification mixture. For example, the presence of RNAase H, which degrades hybrids between DNA and RNA (the basis of the probe hybridization), degrades the specific hybrids in any attempt to probe the impure amplification mix. Also, the presence of reverse transcriptase would use the probes in the hybridization mixture as primers and remove them from the specific hybridization complex formation reaction. In addition to these potential problems, the buffer contains many compounds which might cause problems both for hybridization and also for the generation of ECL by the specific chemistries involved, for example, high levels of salts MgCl.sub.2, KCl, nucleotides, dithiothreitol, spermidine, dimethyl sulphoxide, glycerol and proteins. This list contains many substances which interfere with other nucleic acid probe methods and would need to be removed to allow these methods to work, thus the present invention was surprisingly capable of carrying out a nucleic acid probe assay with such a simple protocol.